Cloning and characterization of SmeDEF, a novel multidrug efflux pump from Stenotrophomonas maltophilia
Cloning and characterization of SmeDEF, a novel multidrug efflux pump from Stenotrophomonas maltophilia
复制标题
DOI：
10.1128/aac.44.11.3079-3086.2000
复制
发表时间：
2000-11-01
影响因子：
4.9
通讯作者：
Martínez, JL
中科院分区：
文献类型：
--
作者：
Alonso, A;Martínez, JL
嗜麦芽窄食单胞菌是一种对多种抗生素具有内在耐药性的医院内致病菌。这种内在的多重耐药表型的机制知之甚少。一个自发性多药耐药沙门氏菌的染色体DNA文库。嗜麦芽窄食单胞菌D457 R突变体（A，Alonso和J.L. Martinet，Antimicrob. Agents Chemother，41：1140-1142，1997）对红霉素敏感性的互补作用进行筛选。克隆和进一步的分析表明，一个6 kbp的区域，构成一个转录单位，能够互补的过敏表型的E。coli Delta acrAB菌株。我们确定了三个开放的阅读框架，smeD，smeE和smeF，分别编码膜融合蛋白，耐药细胞分裂和外膜因子家族的成员。药敏试验表明，在大肠杆菌中克隆的SmeDEF系统具有良好的耐药性。大肠杆菌介导对多种抗生素的耐药性。溴化铵和诺氟沙星积累实验中的存在和不存在的羰基氰间氯苯腙表明，该系统构成了依赖于膜质子动力的药物外排泵。多重耐药D457 R突变体中高水平的smeDEF mRNA的存在与在相同菌株中观察到的高水平的SmeF（以前为Omp 54）一致。相比之下，D457菌株中smeDEF的转录水平很小，这与该菌株中观察到的低水平SmeF相关。此外，对于D457和D457 R菌株，我们观察到生长期依赖性调节，其中最高水平的转录对应于早期指数期，转录在整个生长曲线中降低至24 h时检测不到的水平。
Stenotrophomonas maltophilia is a nosocomial bacterial pathogen intrinsically resistant to several antibiotics. The mechanisms involved in this intrinsic multiresistance phenotype are poorly understood. A library of chromosomal DNA from a spontaneous multidrug-resistant S. maltophilia D457R mutant (A, Alonso and J. L. Martinet, Antimicrob. Agents Chemother, 41:1140-1142, 1997) was screened for complementation of erythromycin susceptibility on an antibiotic-hypersusceptible Escherichia coli Delta acrAB strain. Cloning and further analysis revealed that a 6-kbp region constituting a transcriptional unit was capable of complementing the antibiotic-susceptible phenotype of an E. coli Delta acrAB strain. We identified three open reading frames, smeD, smeE and smeF, which code for members of the membrane fusion protein, resistance nodulation division, and outer membrane factor families, respectively. Drug susceptibility assays indicated that the SmeDEF system cloned in E. coli mediates resistance to a wide range of antibiotics. Ethidium bromide and norfloxacin accumulation experiments in the presence and in the absence of carbonyl cyanide m-chlorophenylhydrazone showed that this system constitutes a drug efflux pump dependent on the membrane proton motive force. The presence of high levels of smeDEF mRNA in the multiresistant D457R mutant was consistent with the high levels of SmeF (formerly Omp54) observed in the same strain. In contrast, transcription levels of smeDEF in the D457 strain were tiny, which correlates with the low levels of SmeF observed for this strain. Also, for both the D457 and D457R strains, we observed growth phase-dependent regulation in which the highest level of transcription corresponded to early exponential phase, with transcription decreasing throughout the growth curve to undetectable levels at 24 h.